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First published online November 16, 2007; 10.1104/pp.107.106229 Plant Physiology 146:888-903 (2008) © 2008 American Society of Plant Biologists OPEN ACCESS ARTICLE
Functional Analysis of the Kunitz Trypsin Inhibitor Family in Poplar Reveals Biochemical Diversity and Multiplicity in Defense against Herbivores1,[C],[W],[OA]Centre for Forest Biology and Department of Biology, University of Victoria, Victoria, British Columbia, Canada V8W 3N5
We investigated the functional and biochemical variability of Kunitz trypsin inhibitor (KTI) genes of Populus trichocarpa x Populus deltoides. Phylogenetic analysis, expressed sequence tag databases, and western-blot analysis confirmed that these genes belong to a large and diverse gene family with complex expression patterns. Five wound- and herbivore-induced genes representing the diversity of the KTI gene family were selected for functional analysis and shown to produce active KTI proteins in Escherichia coli. These recombinant KTI proteins were all biochemically distinct and showed clear differences in efficacy against trypsin-, chymotrypsin-, and elastase-type proteases, suggesting functional specialization of different members of this gene family. The in vitro stability of the KTIs in the presence of reducing agents and elevated temperature also varied widely, emphasizing the biochemical differences of these proteins. Significantly, the properties of the recombinant KTI proteins were not predictable from primary amino acid sequence data. Proteases in midgut extracts of Malacosoma disstria, a lepidopteran pest of Populus, were strongly inhibited by at least two of the KTI gene products. This study suggests that the large diversity in the poplar (Populus spp.) KTI family is important for biochemical and functional specialization, which may be important in the maintenance of pest resistance in long-lived plants such as poplar.
Plants actively respond to challenge by insect herbivores with rapid induction of a suite of biochemical defenses. These defenses can include both secondary metabolites and proteins, which may act as toxins, antifeedants, or antinutrients. One of the most common inducible herbivore defenses in plants is the rapid synthesis of proteinase inhibitors (PIs), small proteins that inhibit insect digestive proteases and lead to reduced insect growth rates or increased mortality (Ryan, 1990  ). Other induced antinutritive proteins of plants include oxidative enzymes that destroy or modify dietary amino acids and fatty acids, such as polyphenol oxidase, peroxidase, and lipoxygenase (Duffey and Felton, 1991), as well as the essential amino acid-degrading enzymes arginase and Thr deaminase (Chen et al., 2005, 2007). Defensive proteins that affect insect growth via other mechanisms include Cys proteases that permeabilize the insect peritrophic matrix (Pechan et al., 2002; Konno et al., 2004; Mohan et al., 2006). Some plants also enhance insect resistance by increasing the synthesis of alkaloids, terpenoids, glucosinolates, and phenolics in response to damage (Walling, 2000; Kessler and Baldwin, 2002). The types of secondary metabolites and proteins with potential antiherbivore activity are numerous and often taxon specific; consequently, plants can affect insects via many different mechanisms (for review, see Constabel, 1999).
PIs are widespread in the plant kingdom and have been described from many plant species and tissues. PIs comprise at least 10 distinct protein families classified by their amino acid sequence and the mechanistic class of proteinases they inhibit (Laskowski and Kato, 1980
As a group, plant KTIs have extremely diverse protease targets and thus have negative effects on a broad range of phytophagous pests and pathogens. Individual KTIs typically have more specific activities. Whereas many plant KTIs inhibit trypsin or chymotrypsin, some can inhibit other Ser proteinases, such as elastase and subtilisin (Terada et al., 1994
Poplar (Populus spp.) has been developed as a woody plant model in genomics and molecular biology and is especially useful as an experimental system for ecological genomics. As trees, poplars are long-lived organisms faced with many generations of pest insects and pathogens. The Populus trichocarpa genome is now fully sequenced (Tuskan et al., 2006 Here we characterize the protease inhibitory activities of five representative wound-inducible members of the KTI gene family as recombinant proteins from Escherichia coli. We determine that all the poplar KTI genes tested encode functional inhibitors, but with different inhibitor profiles, protease preferences, and stabilities. The combined expression patterns and inhibitory activities of the KTIs correlated with protease inhibitor activity of wounded poplar leaf extracts. We also demonstrate inhibitory activity of several KTIs against larval midgut proteases from bertha armyworm (BAW; Mamestra configurata) and forest tent caterpillar (FTC; Malacosoma disstria), pests of Populus and crucifers, respectively. Therefore, our study provides direct evidence of a role of the poplar KTI gene family in the inducible defense of poplar against insect herbivores.
The Poplar KTI Family Contains Many Diverse Members
As part of our ongoing analysis of the poplar defense response, we sought to characterize herbivore- and wound-inducible KTIs in detail. Wounding leaves induces transcript accumulation of several poplar KTIs, including win3 in hybrid poplar and the win3 orthologs PtTI1 and PtTI2 in P. tremuloides (Bradshaw et al., 1990
The three previously identified wound-inducible KTIs (PtdTI3, PtdTI4, and PtdTI5) each belong to separate homology subgroups (Christopher et al., 2004 ; Major and Constabel, 2006). TI3 forms subgroup A3 with 739063, whereas TI4 is a member of group B with another three genes. TI5 does not fit with any of the homology groups and is found on a separate branch. The two recently reported KTIs, TI6 and TI7, belong to subgroup A2, along with two other genes (Talyzina and Ingvarsson, 2006). Group C includes eight genes, none of which have been previously studied.
Inspection of sequence alignments of representative KTIs from each clade/subgroup further illustrates their sequence diversity (Fig. 2
). This made aligning the sequences somewhat unreliable; to improve the quality of the alignment, secondary and tertiary structure predictions were made using JPred, SWISS-MODEL, CPHmodels, and ESyPred3D, which were used to manually edit and refine the alignment. We included the extensively studied soybean (Glycine max) KTI (GmKTI) and sporamin from Ipomoea batatas for comparison. Whereas all KTIs contain the Kunitz motif, they may otherwise share little overall amino acid similarity, including the position of gaps. However, it is interesting that the most conserved regions correspond to predicted β-sheets and, for poplar KTIs, the signal sequence. This is consistent with a recent analysis of molecular evolution of the poplar KTI group A, which showed that the loop regions connecting β-strands, in particular, are under positive selection (Talyzina and Ingvarsson, 2006
Poplar KTIs typically contain four Cys residues that form two conserved intramolecular disulfide bonds. An exception is TI5, which has only two Cys residues that form the first disulfide bond. All poplar KTIs, except TI5 and 554072, also have two additional free Cys residues located in a loop (Cys-165 and Cys-167, win3 numbering). Overall, poplar KTIs share very little similarity with GmKTI (<25% similarity), sporamin, and other well-characterized plant KTIs. This sequence diversity, especially of the reactive loop, predicted that poplar KTIs should have distinct inhibitory activity (see below).
To determine in which tissues the various KTI genes are expressed, we queried PopulusDB for digital expression profiles derived from the abundance of ESTs in diverse cDNA libraries (Sterky et al., 2004
To confirm that the observed wound-induced changes in transcript levels translate into increased protein accumulation, we performed western-blot analysis using the two antibodies available for KTIs. The TI2 antibody is specific for KTIs in the win3/TI2 subgroup and detected several isoforms in leaves (Fig. 4 ). For simplicity, we collectively refer to these bands as TI2. The presence of multiple TI2 isoforms is consistent with the complexity and multiplicity of this subgroup (Bradshaw et al., 1990 ; Hollick and Gordon, 1993; Haruta et al., 2001). By contrast, on western blots the TI3 antibody recognized only a single band corresponding to TI3 protein (Fig. 4). A survey of hybrid poplar tissues determined that TI2-like proteins were found only in wounded leaves, whereas TI3 was present constitutively in most organs, including leaves, bark, wood, and roots (Fig. 4A). TI2 and TI3 were both constitutively present in female catkins of field-grown trees, with minor levels of both proteins in male catkins (Fig. 4B). TI2 protein accumulation in leaves was strongly induced by wounding and TI3 protein showed only modest wound induction. Furthermore, TI2 protein induction was clearly stronger in younger leaves (i.e. at leaf plastochron index [LPI] 3–5), whereas TI3 protein accumulation was relatively consistent independent of tissue age. These KTI proteins thus show very distinct patterns of protein accumulation, but both patterns were consistent with digital northern data (Fig. 3). After wounding, foliar levels of TI2 and TI3 proteins accumulated rapidly after wounding and with similar kinetics; no increase in protein was visible in the first 24 h, but both proteins accumulated to substantial levels 2 d after wounding (Fig. 4C). A similar pattern of TI2 protein accumulation was previously observed in P. tremuloides (Haruta et al., 2001).
Because both transcript and protein levels of poplar KTIs increased in leaves in response to leaf damage, PI activity should also increase. We therefore assayed inhibition of Ser proteinases by crude leaf extracts using a kinetic assay (Worthington, 1988 ); such assays are more accurate than end point assays because activity measurements are effectively the means of continuous time points. Different amounts of leaf extract were tested with trypsin, chymotrypsin, and elastase, three common digestive enzymes found in lepidopteran pests. We first compared TI activity of leaf extracts from immature leaves at LPI 3 to 5 and mature leaves of LPI 9 to 11 and found that the activity was greatest for younger leaves of both untreated and wounded saplings (data not shown). We therefore focused on young leaves (LPI 3–5) for subsequent tests of inhibitory activity against other proteases, and calculated the 50% inhibitory concentrations (IC50) for control and wounded leaf extracts against the three proteases. For undamaged saplings, leaf chymotrypsin inhibition was higher than trypsin inhibition (IC50 of 0.072 g L–1 and 0.23 g L–1, respectively; see Fig. 5
, insets), but wounded leaves contained similar levels of trypsin and chymotrypsin inhibitor activity (IC50 of 0.039 g L–1 and 0.029 g L–1, respectively). Elastase inhibitor activity represented only a small fraction of total inhibitory activity. All three proteinase inhibitory activities increased significantly following wounding (2.5- to 5.9-fold change in IC50; P  0.001). As observed in untreated leaf extracts, elastase inhibitory activity was a small proportion compared to trypsin and chymotrypsin after wound induction. Therefore, as already suggested by western analyses, these data confirm that KTI gene induction is followed by an increase in protease inhibitory activity in poplar leaves.
Recombinant Poplar KTI Proteins Exhibit Distinct Inhibitory Activity and Biochemical Properties
We predicted that the substantial sequence diversity of the poplar KTI genes should translate into diverse biochemical and biological properties of the corresponding proteins. To test this hypothesis, we characterized the functions of TI2, TI3, TI4, TI5, and TI6 proteins in detail because these represent the diversity of the KTI family. High levels of recombinant protein expressed in E. coli were observed for all KTI bacterial expression constructs, with the bulk of the recombinant proteins found in inclusion bodies, as illustrated for TI3 (Fig. 6A
). Proteins were purified under denaturing conditions and dialyzed to renature the polypeptides and recover active protein. We initially tested the renaturation conditions used successfully for TI2 (Haruta et al., 2001
We verified the purity and concentrations of the successfully renatured KTIs by SDS-PAGE, which clearly showed a single major band for each KTI at an approximate predicted Mr (Fig. 6B). In addition, larger bands were apparent for TI3, TI4, and TI6; these may be protein dimers because the apparent Mr of these bands is twice that of the predominant bands. We previously reported putative dimers for TI2 (Haruta et al., 2001 ) and other plant KTIs are known to form dimers as well (Terada et al., 1994). Such dimers may be linked by an intermolecular disulfide bond formed between the free Cys residues of the KTIs (Fig. 2) because the predicted three-dimensional models suggest that residues are exposed on the surface of the proteins (data not shown). Whereas migration of TI2, TI4, and TI6 in gels corresponded well with their predicted Mr (Table I), TI3 and TI5 migrated slower than expected. This is likely the result of residual secondary protein structure because both recombinant proteins migrated according to their predicted Mr on denaturing urea-SDS-PAGE gels, which denature proteins more completely (data not shown). Using in vitro assays with commercially available proteases, we confirmed that the recombinant and renatured poplar KTI proteins have protease inhibitory activity. Commercial GmKTI from soya was included for comparison because it has been extensively described in the literature. Because previous studies of plant KTIs have found a wide range of possible target proteases, we assayed inhibitor activity against several proteases: the Ser proteinases trypsin, chymotrypsin, elastase, and subtilisin, and the Cys proteinase papain. We again used kinetic assays at multiple TI concentrations to test inhibition of trypsin, chymotrypsin, and elastase. End point assays were used to test additional proteinases outside the chymotrypsin subfamily, the Ser protease subtilisin and Cys protease papain. These experiments confirmed that all KTIs tested are active protease inhibitors, but with remarkably different target preferences (Fig. 7 ). Thus, recombinant TI2 was a strong inhibitor of trypsin (Fig. 7, top), with activity levels similar to that of GmKTI, whereas TI3, TI5, and TI6 had intermediate levels of inhibitor activity. In contrast, TI4 did not inhibit trypsin at detectable levels. Against chymotrypsin, TI6 was the most potent of the recombinant poplar KTIs, although less potent than GmKTI, followed by TI3, TI5, and TI4 (Fig. 7, center). Although TI2 was very effective against trypsin, it failed to inhibit chymotrypsin significantly. The only poplar KTI with inhibitory activity against elastase was TI6 (Fig. 7, bottom). None of the poplar KTIs tested was active against subtilisin or papain.
To help compare the inhibitory profiles of the KTIs, we calculated the IC50 for each KTI (Table II ). Such IC50 values permit comparison of inhibitory activities relative to the various proteases tested and emphasize the very different activity profiles of the five poplar KTIs. We note that these are maximal IC50 values because we could not determine directly whether all protein in each preparation was fully refolded. However, we obtained similar inhibitory activity from independent recombinant protein preparations. The comparison of IC50 indicated that several KTIs have strong preferences for specific targets (Table II). TI2 appears to be a specific inhibitor of trypsin, whereas TI4 was specific to chymotrypsin. TI3 and TI5 both inhibited trypsin and chymotrypsin and TI6 inhibited chymotrypsin and elastase. Thus, all the recombinant KTIs tested were active as protease inhibitors and, as a group, inhibited the major digestive enzymes trypsin, chymotrypsin, and elastase.
Recent work has suggested that protein stability is a key function of plant defense proteins given the hostile environment of herbivore digestive systems (Chen et al., 2007 ). We found that KTI activity remained high even after dialysis at pH extremes (Table I). We therefore investigated KTI stability by assessing the denaturing effects of a reducing agent or increasing temperature. Purified recombinant KTIs were first incubated with increasing concentrations of dithiothreitol (DTT) for 30 min, after which remaining inhibitory activity was measured. These activities changed little up to 2 h and thus only data for 30-min incubations are shown (Fig. 8A
). In general, each KTI protein was stable to a critical DTT concentration, beyond which it quickly lost activity. This critical concentration and the rate of activity loss differed for each KTI, giving rise to distinct stability profiles (Fig. 8A). TI4 and TI6 were both resistant to 0.1 mM DTT, but were differentially sensitive at higher concentrations. TI3 retained activity up to 1 mM DTT, but quickly lost activity above this level. TI5 was most resistant to reducing conditions because it retained 65% activity at 100 mM DTT, even after 2-h incubation. Interestingly, TI2 activity increased to an optimal 31.6 µM DTT, but quickly lost activity at higher concentrations and lost all activity beyond 3 mM DTT.
Similarly, we found that poplar KTIs had different stabilities to elevated temperatures. KTI proteins were incubated for 30 min at different temperatures and remaining inhibitory activities were compared with samples kept on ice (Fig. 8B). TI2 was most thermolabile, losing activity above 30°C. Similarly, TI3 was susceptible to temperatures above 40°C. We also repeated these experiments for TI2 and TI3 with gradual cooling after incubation at temperature and found similar loss of activity (data not shown). TI6 was more thermostable than both TI2 and TI3, remaining active after incubation at 60°C. Neither TI2, TI3, nor TI6 were boiling stable because they retained <20% activity after boiling. By contrast, TI5 was stable at all temperatures, retaining 80% to 100% activity at all temperatures assayed. TI4 was also stable at high temperatures, but exhibited enhanced activity of 125% to 130% when incubated at 60°C or greater. In view of the apparent temperature stability of the KTIs, we also measured long-term stability by assaying recombinant KTI preparations at 4°C at 25-d intervals for extended time periods (Supplemental Fig. S1). We found no loss in activity within 150 d, although the measured activity of KTIs fluctuated between 80% and 130%. We extended this analysis to >1 year for TI2, again measuring activity periodically, and found no loss of activity (Supplemental Fig. S1). Overall, these tests demonstrated the remarkable stability of poplar KTI proteins.
The inhibitory activity of recombinant KTIs against commercially available proteases of mammalian origin prompted us to test these KTIs against digestive proteases from insects. We obtained midguts from BAW, a lepidopteran pest of crucifers. The midgut proteases of BAW have been well characterized and, like other lepidopteran larvae, consist primarily of Ser proteinases, particularly trypsin-, chymotrypsin-, and elastase-like enzymes (Hegedus et al., 2003
We also tested poplar KTI inhibition of midgut extracts from FTC, a lepidopteran pest of poplars. We found that protease activity of FTC midguts is due primarily to Ser proteases with a pH optimum of 11.5 (data not shown). As with BAW protease assays, we measured residual protease activity after incubation of poplar KTIs or GmKTI with a standard quantity of FTC midgut extracts. All five KTIs inhibited FTC midgut proteolysis, although only TI2 and TI3 were strong inhibitors (Fig. 10 ). Comparison of inhibition curves revealed that TI3 inhibited FTC gut proteases with comparable apparent potency as GmKTI, and with greater potency than TI2 (Fig. 10). Furthermore, TI3 inhibited almost 70% of protease activity, whereas TI2 inhibited approximately 40%. These maximal inhibition values, which represent the proportion of the mixture of midgut proteases that is susceptible to inhibition, confirmed the potency of these KTIs against FTC and establishes their ecological relevance.
Kunitz TIs have been studied in many different plants and contexts, often with a focus on their potential for biotechnology-based pest control for agriculture; however, the diversity of KTIs within a plant species has not been extensively examined. The prevalence of KTIs in the poplar genome represents a unique opportunity to explore the variation and function of these proteins. Although the precise number of KTI genes in the P. trichocarpa genome has not yet been definitively determined, there could be as many as 30 genes. The region surrounding the win3/TI2 group, in particular, contains much repetitive DNA and appears to have evolved rapidly, making this clade of the gene family difficult to resolve. For functional analysis in the context of plant herbivore defense, we selected five herbivory-inducible genes that represent the diversity of the entire poplar KTI family. Our analysis indicates that each KTI gene product is a functional protease inhibitor with a distinct range of target protease preferences and shows differential stability to reducing agents and temperature.
Based on our interest in herbivory, we chose to characterize representative poplar KTIs from homology groups that were wound or herbivore inducible. Results from published array and northern analyses suggested that group C KTIs generally do not respond to wounding (Fig. 3). Therefore, this group was not included in our analysis, although it is an important target for future study. The inducible nature of TI2 and TI3 was demonstrated at the protein level by western-blot analysis and these proteins showed rapid accumulation after wounding of leaves (Fig. 4). This suggests that, for the other KTIs, induced transcripts are also likely to result in increased levels of the gene products. Furthermore, we showed significant induction of total PI activity in wounded poplar leaves. Interestingly, wounded poplar leaf extracts inhibited chymotrypsin and trypsin almost equally, whereas control leaf extracts were more effective against chymotrypsin. This pattern likely reflects the strong inducibility of TI2 (Fig. 4) and its strong preference for trypsin (Table II). Only low levels of elastase inhibitory activity were found in leaf extracts, as predicted by our finding that only one KTI (TI6) had any significant antielastase activity (Fig. 7). Therefore, total PI activity in leaf extracts generally correlated with the activity of the expressed KTI proteins. Induction of protease inhibitory activity in crude extracts of wounded leaves is important because it demonstrates that up-regulation of KTI genes has a demonstrable impact on herbivore food quality.
Analysis of transcript abundance from digital northerns showed highly diverse expression patterns across the KTI family. For example, based on EST abundance, win3/TI2 expression was highly specific, whereas TI3 showed a very broad pattern of expression, which was confirmed by western analysis (compare Figs. 3 and 4). The win3/TI2 protein accumulated to highest levels in young leaves, consistent with the corresponding transcripts being detected mostly in young leaves, the shoot apex, and floral organs (see below). Most of the other poplar KTIs also exhibited relatively tissue-specific patterns of expression, again with preferred expression in flowers (TI4) and shoot meristem (TI5; Fig. 3). By contrast, TI3 protein and transcripts accumulated in almost all tissues (Figs. 3 and 4). The apparently ubiquitous expression of TI3 is curious because PIs are thought to incur large fitness costs (Zavala et al., 2004
In general, analysis of EST databases predicted that the highest levels of KTI transcripts are present in poplar flowers, particularly female floral buds and catkins. For win3/TI2 and TI3, western analysis confirmed the accumulation of their corresponding proteins (Fig. 4). Hollick and Gordon (1995)
The poplar KTIs tested inhibited trypsin, chymotrypsin, and elastase with different profiles (Fig. 7). TI2 and TI4 had very specific inhibitory activities, being effective against only trypsin or chymotrypsin, respectively. By contrast, TI3 and TI5 inhibited both trypsin and chymotrypsin with similar efficiency. TI6 was the only elastase inhibitor, a rare activity among plant KTIs (Valueva et al., 2000
In poplar, the functional properties of individual KTIs could not be easily predicted based on primary sequence data. For example, although TI2 and TI6 protein sequences were most similar among the KTIs tested (Supplemental Table S1), their inhibitory activity was the most divergent. By contrast, TI3 and TI5 inhibited proteinases with similar potencies, but share little sequence similarity. Likewise, a comparison of reactive site and reactive loop residues revealed that these do not readily predict protease substrate preferences. This may relate to the atypical residues of poplar KTI reactive loops (Fig. 2). Our prediction of KTI reactive loops differs by a window of three amino acid residues from a previous study of Populus group A KTIs (Talyzina and Ingvarsson, 2006
By contrast, the reactive site residues of other types of plant KTIs may be useful indicators of inhibitor specificity. For example, Arg-Lys and Ile-Ser are often reactive site residues in inhibitors of trypsin, whereas Leu-Ser is often the reactive site for KTIs effective against chymotrypsin. Other plant KTIs may have more atypical reactive sites. Glu-Ser are the reactive site residues of DrTI from Delonix regia seeds and sporamin from Ipomoea batatas tubers (Pando et al., 2001
We also observed that the biochemical stability of poplar KTIs differed considerably within the group. Similar to other protease inhibitors, several poplar KTIs retain activity in the presence of the reducing agent DTT and at high temperature (Garcia et al., 2004
In vitro tests with recombinant proteins showed that only TI3 is an active inhibitor of midgut proteases from BAW, a lepidopteran pest of canola (Brassica napus; Fig. 9). Comparison of the initial slopes of the TI3 and GmKTI inhibition curves indicated that TI3 has a stronger affinity for some BAW proteases than soybean GmKTI; however, neither protein completely abolishes proteolytic activity (Fig. 10). When we tested our recombinant KTIs against midgut proteases from FTC, which specialize on P. tremuloides in western Canada, all five KTIs inhibited proteolysis, although TI2 and TI3 were clearly most potent (Fig. 10). The effectiveness of herbivore-induced poplar KTIs on the digestive proteases of a poplar pest strongly supports an important role for KTIs in poplar defense. Furthermore, the differences in effectiveness that we measured against different pests, combined with the differences in target preferences against commercial proteases exhibited for the five KTIs, further support the idea of functional specialization within the poplar KTI gene family.
Compared with Arabidopsis (Arabidopsis thaliana), an annual species, the poplar genome has a larger suite of genes associated with insect resistance (Tuskan et al., 2006
The interaction of poplar KTIs with a diversity of pest protease targets should be reflected in selective pressure on KTI genes. In evolutionary time, this would leave traces of positive selection and rapid molecular evolution in KTI genes in different poplar species or populations. We previously noted the high incidence of restriction fragment polymorphisms and high rates of nonsynonymous substitutions in aspen KTIs (Haruta et al., 2001
Plant Material and Wounding Treatments
Hybrid poplar (Populus trichocarpa x P. deltoids, clone H1-11) plants were propagated and maintained in the Bev Glover Greenhouse at the University of Victoria as described previously (Major and Constabel, 2006
Protein was extracted from leaves of greenhouse-grown poplar by tissue homogenization in sodium phosphate buffer (100 mM, pH 7.0) containing 0.1% (v/v) Triton X-100, 5% (w/v) polyvinylpolypyrrolidone, and 0.1% (v/v) 2-mercaptoethanol. For catkins from field-grown trees, proteins were extracted with Tris buffer (50 mM, pH 7.5) containing 5% (w/v) SDS, 5% (w/v) polyvinylpolypyrrolidone, and 2.5% (v/v) 2-mercaptoethanol. Extracts were clarified by centrifugation, and soluble protein quantified using the Bradford (1976) method for sodium phosphate-buffer extracts or the RC DC protein assay (Bio-Rad) for Tris/SDS-buffer extracts. Proteins were separated by SDS-PAGE and electrotransferred onto polyvinylidene difluoride membranes (Pierce). Ponceau S (Sigma) staining was used to verify equal loading and transfer efficiency. Western-blot detection was carried out using polyclonal antibodies raised against TI2 (Haruta et al., 2001
To obtain the complete poplar KTI family, the P. trichocarpa version 1.0 genome sequence (http://genome.jgi-psf.org/Poptr1/Poptr1.home.html) was queried for gene models annotated as Kunitz-type PIs. KTI gene models are based on the genome version 1.0 annotation because the more recently curated version 1.1 genome sequence (http://genome.jgi-psf.org/Poptr1_1/Poptr1_1.home.html) was missing some KTI gene models that had EST support from GenBank and several models were truncated (data not shown); however, the genome version 1.1 annotations are shown for comparison (Supplemental Table S2). Gene models were combined with previously identified Populus KTI sequences (Bradshaw et al., 1990
Multiple amino acid alignments were made using ClustalW with default parameters, followed by manual adjustments in BioEdit (http://www.mbio.ncsu.edu/BioEdit/bioedit.html). To improve alignments, secondary structure predictions were made using Jpred (http://www.compbio.dundee.ac.uk/
TI2 was expressed and purified as described previously (Haruta et al., 2001
Recombinant proteins were isolated from bacterial inclusion bodies as described previously (Haruta et al., 2001 TI3 antiserum was generated in rats using recombinant TI3 produced as above, but, in addition, we precipitated TI3 protein by dialysis against phosphate-buffered saline at 4°C. Rats were immunized with 100 µg TI3 protein in Freund's complete adjuvant (Sigma), with booster injections of 50 µg protein in Freund's incomplete adjuvant using standard procedures.
All PI activity assays were done in triplicate and the results shown as the means of three replicate assays. The PI activities of recombinant poplar KTIs against trypsin (EC 3.4.21.4; Sigma), chymotrypsin (EC 3.4.21.1; Sigma), and elastase (EC 3.4.21.36; Sigma) were determined by preincubating increasing concentrations of each KTI with a standard quantity of proteinase (6.6 x 10–4 g L–1 final assay concentration) in the appropriate assay buffer. PI activity was determined as described, by measuring residual proteinase activity as the rate of hydrolysis of specific chromogenic substrates (Worthington, 1988 PI activity against subtilisin A (EC 3.4.21.62; Sigma) and papain (EC 3.4.22.2; Sigma) was determined by preincubating increasing concentrations of each KTI with a standard quantity of proteinase in the appropriate assay buffer for 10 min (for subtilisin, 50 mM Tris-HCl, pH 7.5, at 37°C; for papain, 50 mM sodium acetate, pH 6.0, 2 mM DTT at 23°C). After azocasein (Sigma) was added to a final concentration of 1%, the reaction was followed for 1 h and stopped by the addition of TCA. Reactions were clarified by centrifugation, NaOH was added to the supernatant, and the A450 was measured and used to calculate residual proteinase activity.
PI activity of leaf extracts was determined against trypsin, chymotrypsin, and elastase by titrating leaf protein extracts with each proteinase and residual proteinase activity measured as described above. Percent proteinase inhibition was plotted against square root-transformed protein concentration (mg mL–1) of leaf extracts because the relationship between proteinase activity and protein extract was not linear. To directly compare levels of inhibitory activity in leaves, we calculated the total protein concentration of leaf extract, which inhibits 50% of proteinase activity (IC50). For statistical comparison of inhibitor activity in leaves of control and wounding treatments, we compared slopes [% inhibition/(protein extract) For stability tests, recombinant KTIs were incubated under different conditions and, after the denaturing treatment, PI activity was compared with the activity of the untreated KTIs. Inhibition of trypsin was measured for TI2 and TI3, whereas chymotrypsin was used for TI4, TI5, and TI6. For thermostability assays, KTIs were incubated from 10°C to 100°C at 10°C intervals for 30 min, and then cooled on ice for approximately 30 min before measuring residual PI activity. Some thermostability experiments were also performed with gradual cooling after heating, as well as with different incubation times (ranging from 2.5–150 min), but results were the same for both assay variations. To test stability in the presence of a reducing agent, KTIs were incubated with DTT at concentrations increasing exponentially from 1 µM to 100 mM (final concentration) for 30 and 120 min. Reactions were terminated by adding iodoacetamide at twice the concentration of DTT before measuring residual PI activity. For long-term stability, KTIs were stored at 4°C and residual PI activity was measured at 25-d intervals for 100 d. For TI2, residual activity was further measured at 50-d intervals for an additional 200 d.
PI activity against insect proteases was determined from midgut extracts. BAW (Mamestra configurata) extracts were obtained from Dr. Dwayne Hegedus (Agriculture and Agri-Food Canada). Dr. Emma Despland (Concordia University) provided FTC (Malacosoma disstria) midguts dissected from fourth and fifth instar larvae that were reared on artificial diets (Despland and Noseworthy, 2006
The following materials are available in the online version of this article.
We thank Dwayne Hegedus and Emma Despland for providing BAW midgut extracts and FTC midguts, respectively; Steven Ralph and Jörg Bohlmann for providing the TI6 clone (EST ID WS0133_E08; GenBank accession no. DT502517); Charles Melnyk for help with production of active recombinant TI proteins and protease inhibitor assays; Anna Isbister and Lan Tran for help with TI3 antibodies; Nicole Dafoe and Megan Towns for analysis of TI in tent caterpillar frass; and Brad Binges for help with maintaining trees at the Bev Glover Greenhouse. Received August 3, 2007; accepted November 6, 2007; published November 16, 2007.
1 This work was supported by the Natural Sciences and Engineering Research Council of Canada (postgraduate scholarships to I.T.M. and grants to C.P.C.). 
2 Present address: Laurentian Forestry Centre, Canadian Forest Service, Sainte-Foy, Québec, Canada G1V 4C7. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: C. Peter Constabel (cpc{at}uvic.ca).
[C] Some figures in this article are displayed in color online but in black and white in the print edition.
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.107.106229 * Corresponding author; e-mail cpc{at}uvic.ca.
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ABSTRACT
RESULTS
99% identical were considered to be allelic; such sequences typically had synonymous-to-nonsynonymous substitution ratios of 1. For comparison, sequences 
) delineating β-sheets, a boxed region indicating the reactive loop, and asterisks (*) denoting the P1 and P1' reactive site residues. Sequences were retrieved from the National Center for Biotechnology Information with the following accession numbers: GmKTI (
www-jpred
A247 min–1; chymotrypsin activity was assayed with benzoyl-L-Tyr ethyl ester (Sigma) by monitoring the change in 
] from linear regression analysis and calculated a P value (two-tailed) testing the null hypothesis that the slopes are identical (PI activity is equal from control and wound treatments).